Small Ruminant Research 68 (2007) 114–125

Monitoring goat and sheep milk somatic cell counts�

M.J. Paape a,∗, G.R. Wiggans b, D.D. Bannerman a, D.L. Thomas c,A.H. Sanders b, A. Contreras d, P. Moroni e, R.H. Miller b

a Bovine Functional Genomics Laboratory, USDA-ARS, Beltsville, MD 20705, USAb Animal Improvement Programs Laboratory, USDA-ARS, Beltsville, MD 20705, USAc Department of Animal Sciences, University of Wisconsin, Madison, WI 53706, USA

d Animal Health Department, Murcia University, 30071 Murcia, Spaine Department of Animal Pathology, Hygiene and Veterinary Public Health, University of Milano, via Celoria 10, 20133 Milano, Italy

Available online 29 November 2006

Abstract

The milk somatic cell count (MSCC) forms the basis of abnormal milk control programs world wide for goats, cows and sheep.To better understand factors that contribute to elevations in MSCC, the effects of stage of lactation, parity, breed and state/area inthe United States (US) on MSCC were examined. Least squares means were calculated on composite milk somatic cell scores from26,607 goats, 5,944,614 cows and 2197 sheep and the results converted back to MSCC. For goats and cows, MSCC increased withstage of lactation and parity. Counts for cows were lower than counts for goats. By the fifth parity, counts for goats increased to1,150,000 ml−1, exceeding the 1,000,000 ml−1 legal limit for goat milk in the US, whereas maximum counts for cows averagedonly 300,000 ml−1, less than the 750,000 ml−1 legal limit in the US and 400,000 in the European Union (EU). Currently, there isno legal limit for goat milk in the EU. For sheep, MSCC for first parity were higher than for later parities. For later parities, MSCCdecreased with advanced lactation. Cell counts for sheep milk were similar to counts for cow milk. Breed and state/area contributedto variation in cell count for goats and cows. Data were not available for sheep. Studies in the US and EU examined non-infectious

factors contributing to elevations in cell counts. Non-infectious factors such as parity and stage of lactation had minimal effects onMSCC for cows and sheep, but had a major impact on counts for goats, and need to be considered when establishing legal limitsfor goat milk.Published by Elsevier B.V.

at milk

Keywords: Somatic cell count; Abnormal milk; Stage of lactation; Go

1. Introduction

Control of abnormal milk is the most complex andexpensive technical problem facing dairymen today. Theobjective of an abnormal milk control program is to pre-

� This paper is part of the special issue entitled “Goat and SheepMilk” Guest edited by George Haenlein, Young Park, Ketsia Raynal-Ljutovac and Antonio Pirisi.

∗ Corresponding author. Tel.: +1 301 504 8302;fax: +1 301 504 9498.

E-mail address: mpaape@anri.barc.usda.gov (M.J. Paape).

0921-4488/$ – see front matter. Published by Elsevier B.V.doi:10.1016/j.smallrumres.2006.09.014

; Sheep milk; Cow milk; Chemotaxis; Cytokines; PMN neutrophils

vent abnormal milk from entering channels of humanconsumption. The milk somatic cell count (MSCC) isthe basis for abnormal milk control programs for cows,goats and sheep. In the United States (US) the legalMSCC limit established by the Food and Drug Admin-istration for cows is 750,000 ml−1 and for goats andsheep 1,000,000 ml−1. In the European Union (EU)(Directive 92/46ECC Council, 1992) the legal limit for

cows is 400,000 ml−1 and there is no legal limit for goatsand sheep. The MSCC has evolved into an acceptedparameter for the evaluation of milk quality and as amanagement tool for dairymen worldwide. The direct

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SCC was developed to meet the need for a standardizedeference method of determining MSCC in the abnor-al milk control programs being implemented in theS and EU. While MSCC is an accepted procedure

or evaluating cow and sheep milk, it is not applica-le to goat milk. MSCC for uninfected goats appear toe higher than counts for uninfected cows and sheep.actors other than intramammary infection such as man-gement practices, stage of lactation, parity and caprinerthritis-encephalitis virus (CAEV) infection contributeo an elevation of MSCC of goats.

The need for a rapid, accurate and economical methodor large scale counting of somatic cells in both bulkank milk and milk from individual animals has longeen recognized. A method was needed that could bentegrated into laboratories that evaluated large numbersf milk samples. Coulter, Technicon optical, Foss andentley electronic cell counters were developed to fill

his need. Because counting with the Coulter and Tech-icon optical counters required dispersal of fat in theilk and failed to fit into laboratories that also evaluatedilk composition, their use soon lost favor as a means

f determining MSCC. Currently, the Foss and Bentleylectronic cell counters are the industry standards foretermining MSCC.

. Materials and methods

.1. Test-day somatic cell score (SCS)

Data for goats (years 2000–2004) and cows (years 2000–005) on Dairy Herd Improvement test across the US were

ig. 1. Effect of days in milk and lactation number on goat composite milk so= 6989 goats; lactation 3, n = 4617 goats; lactation 4, n = 2990 goats; lactatio

esearch 68 (2007) 114–125 115

submitted to the USDA Animal Improvement Programs Lab-oratory in Beltsville, MD, USA, and a GLM analysis of theSCS (log2[SCC/100,000] + 3) for composite milk samples wasused to estimate SCS means by year, stage of lactation, par-ity, breed and state/region in the US. For sheep, compositeMSCS was obtained during 1997–2004, from a flock main-tained by the Department of Animal Sciences, University ofWisconsin, Madison. The entire data set included compositemilk SCS from 16,041 goats, 3,416,731 cows and 1010 ewes.Least squares means from the SAS GLM procedure were cal-culated on SCS and the results converted back to SCC. Effectsfor year of testing and stage of lactation by parity were esti-mated for all data sets. For goats and cows, breed effects andstate/region in the US also were estimated, and for cows, monthof calving and month of testing effects also were estimated.All effects within species were estimated simultaneously. Alleffects were significant based on the F-statistic.

3. Results and discussion

3.1. Effect of parity and stage of lactation

Results from the above data set indicate that compos-ite MSCC increased with increasing parity and stage oflactation for goats and cows but not for sheep (Figs. 1–3).For goats, counts were lowest at first parity, averag-ing approximately 200,000 ml−1 at 15 days of lactationand reached maximum counts of around 500,000 ml−1

at 285 days of lactation (Fig. 1). By the fifth par-

ity, counts averaged approximately 250,000 ml−1 at 15days and increased to a maximum of approximately1,150,000 ml−1 at 285 days of lactation. While someof the increase in cell counts with increased parity and

matic cell counts (MSCC). Lactation 1, n = 10,130 goats; lactation 2,n 5, n = 1881 goats.

116 M.J. Paape et al. / Small Ruminant Research 68 (2007) 114–125

MSCCactation

Fig. 2. Effect of stage of lactation and lactation number on compositecows; lactation 3, n = 1,103,768 cows; lactation 4, n = 643,404 cows; l

days in milk were probably attributable to increasedintramammary infections, much of the increase was pre-viously attributed to non-infectious factors (Paape andContreras, 1997). Cell counts for uninfected mammaryglands have been reported to increase with stage of lacta-tion and parity (Dulin et al., 1983; Luengo et al., 2004).In the latter study, multiple births and short duration oflactation were also associated with elevated MSCC in

healthy udders. In a recently published study (Moroni etal., 2005), a stage of lactation increase was observed forboth infected and non-infected udders. By 170 days oflactation no difference in MSCC was observed between

Fig. 3. Effect of stage of lactation and lactation number on composite MSCC f3, n = 376 ewes; lactation 4, n = 173 ewes; lactation 5, n = 49 ewes.

for cows. Lactation 1, n = 2,173,447 cows; lactation 2, n = 1,683,5355, n = 341,460 cows.

healthy and infected udders. The mean MSCC for uddersfree from intramammary infection (IMI) was log 3.9(7943 ml−1) and from infected udders it was log 5.6(398,107 ml−1).

In one study, it was determined that more than 90%of the variation in MSCC in goats was not due toIMI (Wilson et al., 1995). They reported that increas-ing days in milk and month of the year were among

the most important factors contributing to increased cellcount in the absence of IMI. To a lesser extent, parityand reduced milk production also contributed signifi-cantly to increased cell count. Interestingly, 75% of the

or ewes. Lactation 1, n = 877 ewes; lactation 2, n = 722 ewes; lactation

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ariation was unexplained. The unexplained variationould be due to infections by Mycoplasma, anaerobicacteria or even CAEV (Paape et al., 2001). Clinicalntramammary infections by Mycoplasma is one of the

ost important causes of elevated MSCC in goat milkCorrales et al., 2004), but the prevalence in the USs low (Wilson et al., 1995). On the other hand, theffect of CAEV on increased MSCC is low (Sanchezt al., 2001; Lerondelle et al., 1992), and similar toSCC produced by coagulase-negative Staphylococci.

n Murciano-Granadina goats free of CAEV and IMI,anchez et al. (1998) showed a progressive increase ineometric mean MSCC from first (140 × 103 cell/ml) tofth lactation (600 × 103 cell/ml).

The increase in MSCC for stage of lactation, seasonnd milk yield were associated with increasing par-ty. Most authors point out that the increase in MSCChroughout lactation could be explained by a dilutionffect, because milk production decreases with increas-ng stage of lactation, and MSCC follows a linearncrease throughout lactation. In addition, when date ofarturition and stage of lactation are similar, the effectf season on MSCC was shown to be related to milkroduction (Sanchez et al., 1998). More recently, milk-ng frequency (1× versus 2×) was shown to have noffect on MSCC over three parities (Salama et al., 2003).cDougall and Voermans (2002) reported that estrus

esulted in increased MSCC, and was independent ofnfection status and milk yield. The influence of non-nfectious factors on bulk tank MSCC for goats makest difficult for goat dairymen to maintain MSCC belowhe US legal limit of 1,000,000 ml−1 (Droke et al., 1993;aenlein and Hinckley, 1995). These factors need to be

onsidered when establishing the legal limit in the EUCorrales et al., 2004; Luengo et al., 2004).

In the USDA data, composite MSCC for cows wereonsiderably lower than counts for goats. In the firstarity at 15 days after parturition, counts averagedpproximately 110,000 ml−1, decreased to approxi-ately 70,000 ml−1 at 45 days and then gradually

ncreased throughout lactation to around 125,000 ml−1

t 450 days (Fig. 2). By the fifth parity counts at day 15ncreased to approximately 170,000 ml−1 and reached

aximum counts of around 300,000 ml−1 at day 450 ofactation. For all five parities, lowest cell counts werebserved at around 45 days of lactation. This was prob-bly attributed in part to a dilution of the cells in milkith increased milk production as the cows approached

eak lactation.

The high counts during early lactation could bettributed to a carry over of the high MSCC in colostrum,eported to be around 2000 × 103 ml−1 (McDonald and

esearch 68 (2007) 114–125 117

Anderson, 1981). The observed increase in cell countsafter 45 days for all parities was probably attributed tothe increased rate of intramammary infections. Regard-less of intramammary infection status, MSCC are higherduring the first few weeks after calving when com-pared to other stages of lactation (Sheldrake et al., 1983;McDonald and Anderson, 1981; Miller et al., 1986).The MSCC for cows has been reported to increase withstage of lactation and lactation number for infected mam-mary quarters but not for uninfected quarters (Paape etal., 1979; Eberhart et al., 1979; Jaartsveld et al., 1983;Sheldrake et al., 1983; Ostensson, 1993). Within a givenlactation, cell counts for uninfected quarters increasedonly 20 × 103 to 80 × 103 cells/ml (Sheldrake et al.,1983; Ostensson, 1993). The stable MSCC during thelarge decrease in milk yield in late lactation suggeststhat the cell content of milk from uninfected quartersis unrelated to milk yield. This is further supported bythe observation that the MSCC for morning and eveningmilkings rarely differed despite large fluctuations in milkyield (Natzke et al., 1972; Paape et al., 1979).

Interestingly, for the sheep flock studied, with theexception of ewes in their fifth parity where counts sud-denly increased at 135 days of lactation, counts didnot increase with parity (Fig. 3). This increase may bedue to the small number (n = 49) of ewes in this par-ity group, where a sudden increase in MSCC for someewes would less likely get diluted out with low cellcount milk. The increase with stage of lactation wasless in first lactation ewes. In later parities, compos-ite MSCC decreased slightly in the second month andthen increased. Similar results were also reported byOthmane et al. (2002). They reported non-significanteffects of stage of lactation and parity on MSCC. Theyattributed this to the strict mastitis control proceduresused in that study. The geometric mean of compositeMSCC of sheep uninfected mammary glands reported byothers averaged <100 × 103 ml−1 and was very similarto that of dairy cows (Marco, 1994; Gonzalez-Rodriguezet al., 1995; Romeo et al., 1996). Pengov (2001) reportedthat 64% of samples from 366 uninfected udder halveshad MSCC less that 50 × 103 ml−1, 81.9% had countsless than 250 × 103 ml−1 and 91.1% had counts less that500 × 103 ml−1. With the exception of 1-year-old ewes,Suarez et al. (2002) reported no effect of age or parityon MSCC. Rupp et al. (2003), however, reported no dif-ference in MSCC in the first (log 3.13 [1349 ml−1]) andsecond lactations (log 3.15 [1413 ml−1]).

Various non-infectious factors have been associatedwith increased cell counts in sheep milk. The most sig-nificant are parity, stage of lactation, time of year, herd,handling of ewes and diurnal variation (Gonzalo et al.,

118 M.J. Paape et al. / Small Ruminant Research 68 (2007) 114–125

site MS527 goa

Fig. 4. Effect of year of Dairy Herd Improvement test date on compoyear 2002, n = 5298 goats; year 2003, n = 5139 goats; year 2004, n = 4

1994a,b; Gonzalo and San Primitivo, 1998). For unin-fected mammary glands, MSCC are highest on the dayof parturition (596 × 103 cells/ml) and decrease duringthe transition from colostrum to true milk, averaging239 × 103 and 186 × 103 ml−1 at 5 and 12 days of lac-tation. Counts continue to decrease with increasing milkproduction and reach minimum values of approximately30 × 103 ml−1 at the fifth week of lactation, which coin-cides with maximum milk production. Counts remainedunchanged for the remainder of the lactation. The changein MSCC throughout lactation was studied by Romeoet al. (1996) at monthly intervals on 799 ewes of theLatxa breed. Uninfected mammary halves (n = 579) hadmean MSCC of 185 × 103 ml−1 and never exceeded477 × 103 ml−1. Infected mammary halves (n = 92) aver-aged 1.5 × 106 ml−1, and never fell below 1 × 106 ml−1.Ewes (n = 128) with mammary halves that were intermit-tently infected averaged 576 × 103 ml−1. The number oflambs delivered at lambing does not influence the MSCCcount (Gonzalo et al., 1994b). For the majority of milkproducing breeds, there is seasonal breeding with mostewes lambing in the winter months. Thus, the seasonalincrease in sheep MSCC reported by others appears tobe linked to the normal lactation curve, where milk pro-duction is lowest during summer and winter months.In that study, a 4–11% increase in MSCC occurredbetween the first and fourth lactation. The diurnal and

daily variations between milkings are similar to thoseobserved in dairy cows. Gonzalo et al. (1994a,b) reporteda 70% increase in the MSCC one hour immediately aftermilking. Other non-systematic factors contributing to

CC for goats. Year 2000, n = 5923 goats; year 2001, n = 5720 goats;ts. Mean (±standard error) MSCC are shown.

variation in MSCC of ewes, such as changes in feeding,have not been studied.

3.2. Yearly trends

In the USDA data, interesting yearly trends wereobserved (Figs. 4–6). For goats, composite MSCCshowed a modest increase over time (Fig. 4). Countsincreased from 525,000 ml−1 in the year 2000 to570,000 ml−1 in 2004. This was surprising because ofthe recent emphasis to lower the legal limit for goatMSCC in the US from 1,000,000 ml−1 to 750,000 ml−1.Thus, one would expect a tendency for the cell count todecrease, not increase, over time. There is no set limitin the EU. For cow milk, composite MSCC averaged142,000 ml−1 in the year 2000 and decreased to 120,000in 2005 (Fig. 5). The legal limit for cell counts for bulktank cow milk in the US is 750,000 and 400,000 ml−1 inthe EU. A recent proposal made by the National MastitisCouncil in the US to lower the MSCC regulatory limit to400,000 by the year 2012 was rejected by delegates to theNational Conference on Interstate Milk Shipments. Forsheep milk, composite MSCC tended to be more vari-able, and increased slightly from 85,000 ml−1 in 1997 to105,000 ml−1 in 2004 (Fig. 6).

3.3. Effect of breed

Breed differences were also observed in the USDAdata for goats and cows (Figs. 7 and 8). Among thegoat breeds, Oberhasli had the lowest cell counts

M.J. Paape et al. / Small Ruminant Research 68 (2007) 114–125 119

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ig. 5. Effect of year of Dairy Herd Improvement test date on composows; year 2002, n = 1,143,085 cows; year 2004, n = 1,162,478 cows;

400,000 ml−1) and Toggenburg the highest (650,000l−1) (Fig. 7). For cows, smaller differences existed

mong breeds when compared to goats (Fig. 8). Milkinghorthorns had the lowest cell counts (125,000 ml−1)nd Guernseys the highest (145,000 ml−1). Breed dif-erences for goats and cows could be attributed to

ifferences in intramammary infection, milk yield orenetics. Data were not available for the sheep breeds.owever, in other studies (Gonzalo et al., 2005), breedifferences on log bulk tank MSCC were observed.

ig. 6. Effect of year of Dairy Herd Improvement test date on composite MS999, n = 242 ewes; year 2000, n = 277 ewes; year 2001, n = 282 ewes; year 2ean (±standard error) MSCC are shown.

C for cows. Year 2000, n = 1,111,956 cows; year 2001, n = 1,137,60305, n = 242,375 cows. Mean (±standard error) MSCC are shown.

Counts ranged from log 5.84 (691,831 ml−1) (Castel-lana breed) to log 6.09 (1,230,269 ml−1) (Awassi andSpanish Assaf), and supported results reported earlierby Gonzalez-Rodriguez et al. (1995).

3.4. State and regional differences

In the USDA data, significant state/regional dif-ferences were observed for goat and cow MSCC(Figs. 9 and 10). For goats, counts were greater than

CC for ewes. Year 1997, n = 201 ewes; year 1998, n = 237 ewes; year002, n = 353 ewes; year 2003, n = 308 ewes, year 2004, n = 297 ewes.

120 M.J. Paape et al. / Small Ruminant Research 68 (2007) 114–125

53 goatgenburg

Fig. 7. Effect of breed on composite MSCC for goats. Alpine, n = 96n = 2851 goats; Nubian, n = 4819 goats; Oberhasli, n = 859 goats; Tog

600,000 ml−1 for Wisconsin, MN, USA, and the South-west region, between 500,000 and 600,000 ml−1 forPennsylvania and Ohio, and between 400,000 and500,000 ml−1 for New York, Iowa, Maryland, Oregonand California (Fig. 9). It would appear that heat stressdid not contribute to elevated MSCC in goat milk,because the Southwest region had counts comparableto Wisconsin and Minnesota. For California, most of the

goat herds were located in Northern California, wherethe temperature is more moderate compared to South-ern California (personal communication, Bill VerBoort,General Manager, California DHIA, Clovis, CA 93612,

Fig. 8. Effect of breed on composite MSCC for cows. Ayrshire, n = 139,243Holstein, n = 50,149,035 cows; Jersey, n = 3,164,043 cows; Milking Shorthornerror) MSCC are shown.

s; Saanen, n = 4619 goats; Experimental, n = 1165 goats; LaMancha,, n = 2641 goats. Mean (±standard error) MSCC are shown.

USA). These data suggest that environmental tempera-ture had little effect on goat MSCC, and that other factorscontributed to the state and regional differences. Resultsfrom other studies also reported no effect of environmen-tal temperature on goat MSCC (Sanchez et al., 1998).

An environmental effect was observed for cow MSCC(Fig. 10). Counts for the Southeast that included stateslike Florida, Georgia, Mississippi and Louisiana were

greater than counts for New York, Pennsylvania, Wis-consin and California. Interestingly, most of the largedairy herds are located in Southern California whereenvironmental temperatures are comparable to states in

cows; Brown Swiss, n = 315,895 cows; Guernsey, n = 227,794 cows;, n = 56,640 cows; Red and White, n = 110,125 cows. Mean (±standard

M.J. Paape et al. / Small Ruminant Research 68 (2007) 114–125 121

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ig. 9. Herd test-day composite MSCC for goats by state and regionoats; OH, n = 2929 goats; WI, n = 3092 goats; MN, n = 3602 goats;A, n = 4708 goats. Mean (±standard error) MSCC are shown.

he Southeast (personal communication, Bill VerBoort,eneral Manager, California DHIA, Clovis). This would

uggest that perhaps humidity may have contributed tohe increased counts. Also, mastitis control programsend to be less vigorous for small herds when comparedo large herds (Norman et al., 2000).

.5. Differential MSCC

Unlike cow milk where macrophages are the pre-ominant cell type (Ostensson et al., 1988; Ostensson,

ig. 10. Herd test-day composite MSCC for cows by state and region of the UnI, n = 1,446,846 cows; Southeast, n = 317,174 cows; CA, n = 2,362,952 cow

United States during 2000–2004. NY, n = 3465 goats; PA, n = 1582414; MD, n = 1173; Southwest, n = 1148 goats; OR, n = 3494 goats;

1993; Miller et al., 1986), polymorphonuclear neu-trophils (PMN) comprise the major cell type in milkfrom infected and uninfected mammary glands of goats(Dulin et al., 1983). For animals free of intramam-mary infection, PMN constitute 45–74% of the somaticcells in goat milk and 71–86% for infected mammaryhalves. Macrophages comprise 15–41% of the somatic

cells in uninfected halves and 8–18% in infected halves.Lymphocytes comprise 9–20% of the somatic cells inuninfected halves and 5–11% in infected halves. Epithe-lial cells are low in goat milk, but identification by light

ited States during 2000–2005. NY, n = 853,103 cows; PA, n = 964,539;s. Mean (±standard error) MSCC are shown.

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microscopy is difficult because of the presence of cyto-plasmic particles in goat milk. An early study reportedthat epithelial cells comprised less than 1% of the totalcells. More recent studies reported that 6% of the cellsin uninfected mammary halves were epithelial in origin(Contreras, 1998). Because milk secretion in the goat isapocrine (Wooding et al., 1970), cytoplasmic particlesare shed into milk from the apical portion of mammarysecretory cells. The numbers of cytoplasmic particlesin milk of uninfected mammary halves range from 71to 306 × 103 ml−1 and for infected mammary halvesfrom 98 to 231 × 103 ml−1 (Dulin et al., 1983). Althoughthe majority of these particles are generally anucleated,approximately 1% contain nuclear fragments (Dulin etal., 1982).

Limited data exist on changes in differential MSCCthroughout lactation for ewes. Similar to dairy cows, themacrophage is the predominant cell type (46–84%) inmilk from uninfected mammary glands of ewes (Cuccuruet al., 1997). The PMN comprise 2–28% of the cellpopulation and lymphocytes (11–20%). Plasma cellsare present in small numbers in colostrum (0–20%),as well as epithelial cells (1–2%). For infected mam-mary glands, the percentage of PMN increases to 50%at a MSCC of 200 × 103 ml−1 and to 90% at a MSCCover 3 × 106 ml−1. Cytoplasmic particles are normalconstituents in ewe milk and colostrum. However, con-centrations are 10 times less than counts in goat milk,averaging 15 × 103 cells/ml (Martinez et al., 1997). Arecent study by Albenzio (2004), reported no significantstage of lactation effect on differential MSCC.

3.6. Mechanisms responsible for increased MSCCduring infection

Increases in MSCC during intramammary infectionare an essential part of the mammary gland’s defensesagainst an invading pathogen. The initial increases inmilk somatic cells are primarily due to the recruitmentof circulating PMN from the circulation to the inflamedtissue (Persson-Waller et al., 1997). Once PMN havemigrated to the gland and become activated, they releasea number of anti-bacterial components that are essentialfor successful host clearance of the infectious pathogen(Paape et al., 2002, 2003).

Recruitment of PMN to the gland occurs through aprocess referred to as chemotaxis (Wagner and Roth,2000). Chemoattractants are soluble molecules secreted

from inflamed tissue which enable directional migrationof PMN to the site of infection. In addition to chemoat-tractants, PMN chemotaxis requires the expression andinteraction of complementary adhesion molecules on

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PMN and endothelial cells, the latter of which line theluminal surface of the vascular wall and regulate leuko-cyte trafficking (Carlos and Harlan, 1994; Wagner andRoth, 2000).

Within the past decade, the mechanisms responsiblefor PMN recruitment, and thus increases in milk SCC,have been elucidated in small ruminants such as sheep.As mentioned previously, surface adhesion moleculesplay a requisite role in PMN adherence to and migra-tion through the endothelial lining of the vascular wall(Carlos and Harlan, 1994; Wagner and Roth, 2000).Similar to other species, ovine PMN express l-selectin(CD62L) and CD18 (Persson-Waller and Colditz, 1998).l-Selectin is constitutively expressed on PMN surfacesand mediates rolling of PMN along the endotheliumlining of post-capillary venules. CD18 mediates firmattachment of PMN to the endothelium and facili-tates PMN transendothelial migration. The intensity ofl-selectin staining is lower on ovine PMN in milk com-pared with those in blood (Persson-Waller and Colditz,1998). In contrast, CD18 staining is higher on milk-derived ovine PMN than on those obtained from blood.The differential surface expression of these two ovineadhesion molecules between pre- and post-migratedPMN is consistent with the shedding of l-selectin and theupregulation of CD18 during transendothelial migrationand is comparable to that seen in other species, includingcattle (Riollet et al., 2000) and humans (Keeney et al.,1993).

Cytokines play a critical role in PMN recruitment toinflamed tissue (Wagner and Roth, 2000). These sol-uble, cell-derived molecules influence cell responses,such as adhesion molecule expression, by binding tocell surface receptors and activating intracellular signaltransduction pathways leading to transcriptional activa-tion. In humans, two well-described pro-inflammatorycytokines, TNF-� and IL-1�, induce vascular endothe-lial adhesion molecule expression, thereby, promotingPMN transendothelial migration to the site of infec-tion (Carlos and Harlan, 1994; Wagner and Roth, 2000).Another cytokine involved in PMN recruitment is IL-8,which is directly chemotactic for PMN (Harada et al.,1994; Wagner and Roth, 2000).

There are data to suggest that the ovine orthologuesof the aforementioned human cytokines have a similarrole in mediating PMN recruitment to infected mammaryglands. Experimental intramammary infection of sheepwith Staphylococcus aureus or Escherichia coli has been

shown to induce a significant increase in milk leukocyteswithin 24 h of infection (Persson-Waller et al., 1997).Similar to cattle (Bannerman et al., 2004), E. coli intra-mammary infection elicited a more rapid recruitment of

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MN to the gland than S. aureus. Interestingly, this delayn PMN recruitment in response to S. aureus in both cat-le and sheep correlated with impaired clearance of S.ureus relative to that of E. coli. Maximal increases inilk levels of ovine TNF-� and IL-8 preceded or were

emporally coincident with maximal PMN recruitment tolands infected with either pathogen (Persson-Waller etl., 1997). Relative to E. coli-infected glands, a delay innduction of peak levels of these cytokines in S. aureus-nfected quarters corresponded with a respective delayn maximal leukocyte recruitment (i.e., elevated milkCC).

In contrast to the induction of comparable concentra-ions of TNF-� and IL-8 in glands infected with eitherathogen, appreciable levels of IL-1� were only detectedn S. aureus-infected glands. Elevations in milk IL-1�evels in S. aureus-infected glands paralleled increasesn milk neutrophils. IL-1� production in sheep is notnly elicited by S. aureus, as intramammary infectiony another Gram-positive bacterium, Staphylococcuspidermidis, has similarly been reported to elicit itsroduction (Winter and Colditz, 2002; Winter et al.,003). The minimal induction of IL-1� in response to. coli is consistent with another report demonstrat-

ng negligible production of ovine IL-1� in quartersnfused with endotoxin (Waller et al., 1997), a highlymmunostimulatory component of the cell wall of allram-negative bacteria, including E. coli. Similar to. coli intramammary infection, increases in ovineNF-� and IL-8 were detected in endotoxin-challengeduarters.

That increases in milk levels of TNF-�, IL-8 andL-1� following intramammary infection are temporallyoincident with increases in milk PMN suggests a roleor these cytokines in ovine PMN recruitment. Directvidence supporting this notion has been provided intudies investigating the direct effects of these cytokinesn changes in PMN levels in lactating ovine glandsPersson et al., 1996). Infusion of ovine IL-1� or TNF-�nto either the teat cisterns or udders of sheep inducedn increase in leukocytes, the majority of which wereMN. Infusion of ovine IL-8 into the teat cistern, butot the udder, elicited an increase in PMN. Although IL-has been reported in vitro to be chemotactic for both

aprine (Barber et al., 1999) and ovine PMN (Muldernd Colditz, 1993), the lack of an effect of IL-8 on PMNecruitment when infused into the ovine udder remainsnexplained. One may speculate that a low potency of IL-

combined with the dilutional effect of milk may have

blated its chemotactic activity. Together, the identifi-ation of cytokines and adhesion molecules responsibleor PMN recruitment enhances our understanding of the

esearch 68 (2007) 114–125 123

mechanism by which elevations in MSCC occur duringintramammary infection.

4. Conclusions

Intramammary infection is the major cause for ele-vated SCC in milk of dairy ruminants. The innateimmune system calls into play a host of cytokines criticalin the early recruitment of PMN to the mammary glandin response to invading mastitis pathogens. The increasein MSCC due to stage of lactation and parity for cowsand sheep are mainly the result of intramammary infec-tions. While intramammary infection increases MSCCfor goats, other non-infectious factors such as estrus, sea-son and milk yield will also increase counts in goat milk.For non-infected goat halves, a progressive increase inMSCC is also observed with parity and advanced lac-tation. North American goat dairymen have difficultiesin maintaining bulk tank MSCC below the threshold of1,000,000 ml−1. There is currently no legal limit for goatmilk in the EU. Non-infectious factors that contributeto elevations in MSCC for goats need to be consideredwhen establishing legal cell count limits.

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